The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the αβ TCR. However, T cells that express the γδ TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ T cell numbers increased 10-fold in the infected ear and resulted in a novel γδ T cell population not found in naive skin. Circulating γδ T cells were specifically recruited to the site of inflammation and differentially contributed to dermal populations based on their CD27 expression. Recruited γδ T cells, the majority of which were CD27+, were granzyme B+ and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident γδ T cell populations that made IL-17 were CD27. Using a double-chimera model that can discriminate between the resident dermal and recruited γδ T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflammation. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and collateral damage in the tissue during viral infection. These findings have important implications for our understanding of immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune defense.

The skin is host to a network of T lymphocytes, some of which take up permanent residence within the tissue, having been recruited there throughout life, whereas others are only transiently present (1, 2). Following Ag-specific activation in central lymphoid organs, TCRαβ effector cells traffic to peripheral sites, including the skin, and some become resident memory T cells. These cells are recruited into the tissue as a result of local infection or inflammatory signals and remain there to protect against future external insults (38). In contrast to TCRαβ resident memory T cells, TCRγδ T cells were shown to home to tissues and acquire specific functions as a result of developmental programming, and their response via TCR recognition of foreign Ags is poorly defined (911). This hardwiring, which drives them particularly to barrier tissues, such as the intestine, reproductive tract, and skin, seems ideally suited to ensure that γδ T cells participate in innate host defense.

Within the murine skin, dendritic epidermal T cells (DETC) are resident in the epidermis. They seed the skin during fetal development, express an invariant Vγ5 TCR, have dendritic morphology, and are relatively immobile (12). They have been shown to participate in keratinocyte maintenance, to respond to wounding, and to assist in the healing process (1315). A different subset of TCRγδ T cells is resident in the dermis. Dermal γδ T cells account for up to half of the dermal T cells in mice (16) and 2–9% in humans (17). This population was shown to participate in host defense against bacterial infection of the skin (16, 18), as well as to contribute to psoriasis in the mouse (19, 20) and humans (21). It is now appreciated that, in mice, dermal γδ T cells exit the thymus and seed the dermis near the time of birth (11, 22). Characterization of dermal γδ T cells by several groups revealed their memory phenotype and functions related to those of CD4+ Th17 cells, being capable of producing IL-17 upon stimulation in vitro. Dermal γδ T cells express CCR6, CXCR6, CD103, CD44, IL-23R, and IL-7R (2123); however, it is unclear whether IL-17–producing dermal γδ T cell populations observed during infection or inflammation are derived from the resident population having been seeded there at birth or whether unique populations are recruited from the circulation.

A recent study identified a population of γδ T cells that is enriched in skin draining lymph nodes (dLN) of mice with properties similar to those of dermal γδ T cells (22). This discovery demonstrated a potential link between dermal γδ T cells and those found in the circulation. Although several models of systemic viral infection (24, 25) elicited a γδ T cell response in the spleen and lymph nodes (LN), these studies did not address questions concerning tissue trafficking or provide a relevant context for natural modes of infection. Importantly, studies with human PBMC found that γδ T cells in the circulation are impacted in patients with inflammatory bowel disease (26), melanoma (27), or psoriasis (28), suggesting that local inflammation can affect global γδ T cell homeostasis. These findings point to the need to understand the trafficking patterns of γδ T cells during steady-state conditions and in the context of disease.

Emerging data from studies of cells in central lymphoid organs suggest that CD27 delineates γδ T cell functional subsets, leading to a paradigm shift for how γδ T cells are classified (11, 29). Expression of CD27 by mouse γδ T cells is tightly correlated with their ability to produce IFN-γ, where failure to express CD27 correlates with IL-17 production (30). Importantly, analogous γδ T cell populations were found in human PBMC (3133), making this a relevant axis from which to orient experiments in model organisms.

In the current study, we analyzed the γδ T cell response in mouse skin to local infection with vaccinia virus (VV). We sought to dissect the contribution of resident versus immigrant γδ T cells following infection and to determine whether γδ T cells recruited from the circulation give rise to long-term residents in the skin after the cutaneous infection has cleared. Many previous reports studied the γδ T cell response in TCRα−/− or TCRβ−/− mice, which lack TCRαβ T cells (24, 25), or performed transfers of γδ T cells isolated from TCRβ−/− mice (34) to investigate the antiviral response. In such scenarios, the γδ T cell makeup and response are likely abnormal (35). Analyzing γδ T cells in a wild-type (WT) setting allowed us to better appreciate their role in a skin infection. Our experiments revealed that CD27+ and CD27 γδ T cells are specifically recruited from the circulation to the site of infection; surprisingly, however, they do not become resident in the tissue, even when encountering a relatively empty niche. The immigrant γδ T cells have functional attributes distinct from the resident dermal γδ T cells, with the latter being primarily responsible for enhancing the inflammatory response in the tissue.

C57BL/6, B6.SJL-Ptprca Pepcb/BoyJ, and TCRδ−/− mice were obtained from the Jackson Laboratory, and TCRδ-H2BeGFP (TCRδ-GFP) mice were obtained from I. Prinz (Hannover Medical School, Hannover, Germany); all were described previously (36). Mice were housed in specific pathogen–free conditions in the animal facilities at the University of Washington and, unless otherwise specified, were used at 6–10 wk of age. All experiments were done in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Washington.

A total of 2 × 106 PFU recombinant VV expressing full-length chicken OVA was used for epicutaneous infection by skin scarification, as described previously (37). Mice were anesthetized with isoflurane, and 5 μl diluted virus was applied to the dorsal side of the ear. The skin area was gently scratched 20 times with a 28-gauge needle. For flow cytometry experiments, both ears of mice were infected and combined to make up one sample, and the numbers were divided by two to obtain the number of cells/ear.

C57BL/6 or CD45.2+ TCRδ-GFP mice received 7 × 105 γδ T cells enriched from spleen and skin dLN of CD45.1+ TCRδ-GFP mice by i.v. injection 1 d before infection. Enrichment of γδ T cells was performed by incubating single-cell suspensions with biotin-labeled anti-TCRβ and anti-CD19 mAb for 30 min on ice, followed by passage through the Stem Cell Biotin negative selection kit. Mice were infected on one ear, and the infected (or uninfected) ears from two mice were combined for each sample. In some experiments, the enriched population was further purified as follows: cells were stained with fluorochrome-conjugated anti-CD27 and sorted on a FACSAria for the GFP+TCRβCD19CD27+ or GFP+TCRβCD19CD27 population, of which >97% were TCRγδ+. A total of 6 × 105 CD27+ or 2.7 × 105 CD27 sorted γδ T cells from B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice was transferred into separate C57BL/6 (CD45.2) hosts. Mice that received sorted cells were infected on both ears, and ears from a single mouse were combined for a sample.

Single-cell suspensions were prepared from spleens and LN by mechanical disruption. Dorsal and ventral halves of ears were separated, minced, and enzymatically digested for 2 h at 37°C in Liberase TM (Roche). Both ears from each mouse were combined for processing to obtain adequate cell numbers. All tissues were passaged through a 100-μm nylon sieve (BD Bioscience). In some experiments, to avoid the Liberase digestion (that cleaved CD27), dorsal and ventral halves of the ears were separated and floated dermal-side down in complete media overnight at 37°C. Supernatant, into which cells had migrated, was collected the following day and stained. Cells were stained for 30 min on ice with the appropriate mixture of mAb and washed with PBS containing 1% BSA. The following conjugated mAb were obtained from BD Pharmingen, eBioscience, or BioLegend: anti-TCRβ (H57-597), CCR6 (140706), CD27 (LG.3A10), CD103 (2E7), Vγ5 (BioLegend Vγ3 clone 536), CD44 (IM7), CD45.1 (A20), and CD45.2 (104). In the ear, γδ T cells from TCRδ-GFP were identified as GFP+Vγ5 (dermal) or GFP+Vγ5+ (DETC), according to the nomenclature described by Heilig and Tonegawa (38). For the analysis of intracellular IFN-γ, IL-17, and granzyme B, single-cell suspensions were incubated in the presence or absence of 50 ng/ml PMA and 500 ng/ml ionomycin for 4 h at 37°C in the presence of brefeldin A. Cells were stained for surface markers and then processed using the BD Pharmingen Cytofix/Cytoperm kit. Samples were analyzed on a FACSCanto II (BD) using FlowJo software (Tree Star).

Infected mice were injected i.p. with 2 mg BrdU in 200 μl PBS1 h before being euthanized. Single-cell suspensions were obtained from each tissue and stained using the BD Biosciences APC BrdU Kit. Cells were fixed with paraformaldehyde before Cytofix/Cytoperm to ensure retention of the TCRδ-GFP signal.

Chimeras were made as previously described (22). Briefly, perinatal thymocytes (pThy) were harvested 0–48 h after birth, and 5 × 106 cells were transferred i.v. to congenic recipients that had been lethally irradiated with 1000 rad in a cesium irradiator. The next day, 1–2 × 106 congenic bone marrow (BM) cells were transferred. These mice are referred to as “BMpThy chimeras,” whereas controls without pThy are referred to as “BM chimeras.” Chimeras were analyzed or infected ≥8 wk after reconstitution.

The viral load in organs was determined by plaque assays on 143B cells, with dilutions of a 40% tissue homogenate added to confluent wells. Titers reported are log10 PFU per ear.

Ears from infected mice were fixed in 10% buffered formalin for 3 d before being embedded vertically in paraffin. Sections were stained with H&E, and the severity of inflammation and necrosis was determined on a scale of 0–4 (with 4 being the most severe). Scoring of all samples was blinded, and uninfected mice were used as controls.

Prism software (GraphPad) was used for all statistical analysis. The two-tailed, unpaired Student t test was used for comparisons of γδ T cell frequencies and histological scores.

Although it is known that γδ T cells reside at barrier surfaces in mice and humans (16, 17), few studies have addressed the role that they play during infection of the skin, particularly in response to viral infection. To address this, we used a model of VV scarification, in which virus localizes primarily to the site of inoculation (39). Following infection of the ear pinna, we observed that the number of dermal γδ T cells increased ∼10-fold in the infected ear and dLN (Fig. 1A, 1B). The response kinetics were slightly unusual, because the numbers in the skin and dLN remained elevated for a protracted period of time. Furthermore, the number of dermal γδ T cells at late time points was significantly higher than in naive ears, suggesting that infection could result in prolonged alterations in the dynamics of the dermal γδ T cell population. The number of DETC also increased ∼3-fold following infection and returned to the number found in control ears at late time points (Fig. 1A).

FIGURE 1.

Following skin scarification with VV, γδ T cells accumulate in dLN and dermis. TCRδ-GFP mice were infected with VV on the ear by scarification. γδ T cells were gated as TCRβGFP+ in the LN and as TCRβGFP+Vγ5+ (DETC) or TCRβGFP+Vγ5 (dermal γδ T cells) in the ear. (A) Number of DETC and dermal γδ T cells in the ear following infection. Open circle represents the number of dermal γδ T cells in the contralateral uninfected ear at 7 dpi. (B) Number of γδ T cells in the dLN following infection. Open circle represents the number of γδ T cells in the contralateral non-dLN (ndLN) at 7 dpi. (C) Representative flow plots show CCR6 and CD103 profile of dermal γδ T cells following infection. Numbers denote the percentage of the total dermal γδ T cell population within the indicated quadrant. (D) Cell numbers within the indicated dermal γδ T cell subsets/ear over the course of infection (inset, day 0). Error bars signify SEM. Data are compiled from >5 (A and B) or >3 (C and D) independent experiments (n = 3–13 mice/group). *p < 0.001, two-tailed, unpaired t test.

FIGURE 1.

Following skin scarification with VV, γδ T cells accumulate in dLN and dermis. TCRδ-GFP mice were infected with VV on the ear by scarification. γδ T cells were gated as TCRβGFP+ in the LN and as TCRβGFP+Vγ5+ (DETC) or TCRβGFP+Vγ5 (dermal γδ T cells) in the ear. (A) Number of DETC and dermal γδ T cells in the ear following infection. Open circle represents the number of dermal γδ T cells in the contralateral uninfected ear at 7 dpi. (B) Number of γδ T cells in the dLN following infection. Open circle represents the number of γδ T cells in the contralateral non-dLN (ndLN) at 7 dpi. (C) Representative flow plots show CCR6 and CD103 profile of dermal γδ T cells following infection. Numbers denote the percentage of the total dermal γδ T cell population within the indicated quadrant. (D) Cell numbers within the indicated dermal γδ T cell subsets/ear over the course of infection (inset, day 0). Error bars signify SEM. Data are compiled from >5 (A and B) or >3 (C and D) independent experiments (n = 3–13 mice/group). *p < 0.001, two-tailed, unpaired t test.

Close modal

Dermal γδ T cells were reported to express CCR6 and CD103 (22). We confirmed that, in resting skin, all dermal γδ T cells are CD103+, and more than half are CCR6+ (Fig. 1C, 1D). Skin scarification with VV resulted in dramatic changes in the dermal γδ T cell population. By 3 d postinfection (dpi), a novel subset appeared in the dermis that was CCR6 and CD103 double negative (DN). This novel dermal γδ T cell subset accounted for almost half of the γδ T cell population at the peak of the response and then disappeared entirely from the dermis by 30 dpi (Fig. 1C, 1D). We also assessed TCR Vγ-chain expression using commercially available Ab on the three dermal populations and found that all were similarly heterogeneous (data not shown).

We performed an adoptive transfer to investigate the origin of the dermal γδ T cell populations following infection and to determine the contribution of circulating γδ T cells to the expanded dermal population. Accordingly, 7 × 105 γδ T cells isolated from the spleen and skin dLN of naive TCRδ-GFP mice were transferred i.v. into normal C57BL/6 recipients before VV scarification. Infection of the recipients showed that donor γδ T cells were specifically recruited to the skin of the infected ear, and their numbers peaked 7 dpi (Fig. 2A). Although these adoptively transferred, circulating cells were capable of entering the dermis during active infection, very few remained permanently integrated into the dermis at a late time point.

FIGURE 2.

Circulating γδ T cells migrate specifically to VV-infected skin and contribute to all three dermal subsets. C57BL/6 mice (CD45.2) or CD45.2+ TCRδ-GFP mice received 7 × 105 γδ T cells isolated from spleen and skin dLN of CD45.1+ TCRδ-GFP mice. The following day, mice were infected with VV on the ear. (A) Graphs show numbers of transferred TCRδ-GFP+ cells recovered from the LN or ear (data are mean ± SEM). (B) Representative flow plot shows the CCR6 and CD27 profile of the donor γδ T cell population prior to transfer. (C) Representative flow plots show CCR6 and CD103 profile of donor and recipient dermal cells at 7 dpi. (D) γδ T cells from spleen and skin dLN of TCRδ-GFP mice were sorted based on CD27 expression, and 6 × 105 CD27+ or 2.7 × 105 CD27 γδ T cells were transferred into separate C57BL/6 hosts. Recipients were infected with VV on the ear the following day. Flow plots show the phenotype of CD27 and CD27+ at the time of transfer and 7 dpi in the spleen and ear. Numbers denote the percentage of cells within the indicated quadrant. Data are representative of at least two independent experiments (n = 3–7 mice/group).

FIGURE 2.

Circulating γδ T cells migrate specifically to VV-infected skin and contribute to all three dermal subsets. C57BL/6 mice (CD45.2) or CD45.2+ TCRδ-GFP mice received 7 × 105 γδ T cells isolated from spleen and skin dLN of CD45.1+ TCRδ-GFP mice. The following day, mice were infected with VV on the ear. (A) Graphs show numbers of transferred TCRδ-GFP+ cells recovered from the LN or ear (data are mean ± SEM). (B) Representative flow plot shows the CCR6 and CD27 profile of the donor γδ T cell population prior to transfer. (C) Representative flow plots show CCR6 and CD103 profile of donor and recipient dermal cells at 7 dpi. (D) γδ T cells from spleen and skin dLN of TCRδ-GFP mice were sorted based on CD27 expression, and 6 × 105 CD27+ or 2.7 × 105 CD27 γδ T cells were transferred into separate C57BL/6 hosts. Recipients were infected with VV on the ear the following day. Flow plots show the phenotype of CD27 and CD27+ at the time of transfer and 7 dpi in the spleen and ear. Numbers denote the percentage of cells within the indicated quadrant. Data are representative of at least two independent experiments (n = 3–7 mice/group).

Close modal

In the peripheral lymphoid organs and the circulation, it was established that γδ T cells can be functionally segregated based on CD27 expression (11, 29). We found that CD27 and CCR6 staining were mutually exclusive in peripheral lymphoid organs and identified three γδ T cell populations with distinct surface and transcription factor staining patterns (Fig. 2B, Supplemental Fig. 1A). In line with previous reports that CD27 γδ T cells produce IL-17 upon stimulation, we found that baseline expression of the transcription factor RORγt was higher in CD27 than in CD27+ γδ T cells. Conversely, the transcription factor Tbet was highest in the CD27+ γδ T cells. CCR6+CD27 γδ T cells expressed the highest levels of CD44 and CD103, whereas the small number of CD27CCR6 cells and the CD27+ cell subset had lower levels of CD44, and only half expressed CD103.

Transfer experiments revealed that the donor γδ T cells contributed to all three dermal γδ T cell populations defined by CD103 and CCR6 staining (Fig. 2C), but it was not clear how these populations related to those in the circulation. Our initial attempts to stain for CD27 on γδ T cells isolated from the ear were negative. Further analysis revealed that CD27 was cleaved during the digestion process used to isolate cells from the ear. Floating ears (dorsal and ventral sides separated) overnight in complete media without enzymatic digestion released some γδ T cells from the tissue and permitted analysis of CD27 expression. This technique revealed that, although CD27 was not found on dermal γδ T cells in naive animals, it was expressed on some dermal γδ T cells following infection, all of which belonged to the CD103CCR6 DN population (Supplemental Fig. 1B). This suggested that the CD103CCR6 DN subset appearing in infected ears was an immigrant population of circulating CD27+ γδ T cells. Because yields and subset composition from the floating technique were variable, we opted to use CD103 and CCR6 for further analyses of skin samples.

To determine whether CD27+ or CD27 γδ T cells from the circulating pool differentially contributed to the three dermal γδ T cell subsets, we sorted and transferred them into separate hosts before ear scarification with VV. The CD27 donor γδ T cells contributed to all three dermal populations defined by CD103 and CCR6, although few were DN, and most expressed CD103 (Fig. 2D). In contrast, immigrant γδ T cells from the CD27+ donor population were exclusively CD103CCR6 DN, even though 50% of cells in the donor population expressed CD103 (Supplemental Fig. 1A). On a per-cell basis, the CD27 γδ T cells were recruited more efficiently to the infected skin (data not shown). These experiments additionally demonstrate that in vivo in the presence of inflammatory signals, CD27 expression remains stable in the LN and spleen as a marker of distinct γδ T cell lineages. Based on these experiments and the fact that we observed very limited proliferation within the tissue (Supplemental Fig. 2), it appears that the majority of γδ T cell accumulation is due to recruitment.

To tease apart the distinct roles of the resident dermal and circulating γδ T cell populations, we generated mice with and without resident dermal γδ T cells (22). We found that γδ T cells in the circulation and dermis were sensitive to whole-body irradiation and that donor BM failed to reconstitute the dermal (Fig. 3A) or the circulating CCR6+ γδ T cell populations (Supplemental Fig. 3). The majority of the γδ T cells in the spleen and LN derived from the BM were CD27+ with a minority of CD27CCR6 γδ T cells. Although small numbers of host γδ T cells remained in the dermis following irradiation (Fig. 3B), their phenotype was abnormal (data not shown), and their numbers were significantly lower than in unirradiated mice.

FIGURE 3.

Contribution of adult BM-derived and perinatal thymus-derived γδ T cells to the dermal response. CD45.2+ TCRδ-GFP mice were irradiated and reconstituted with BM or with BM plus pThy (BMpThy). (A) Graphs show the total number of γδ T cells >8 wk following reconstitution in the dermis and skin dLN. Geometric mean is shown. (B) Contribution of BM-derived and pThy-derived γδ T cells in the dermis and LN. Geometric mean is shown. (C and D) BMpThy chimeras were infected with VV on the ear >8 wk following reconstitution. (C) CCR6 and CD27 profiles of γδ T cells in the skin dLN at day 0 and CCR6 and CD103 profiles of γδ T cells in the dermis at day 0 and 7 dpi. Numbers denote the percentage of that population within the indicated quadrant. (D) The number of BM- and pThy-derived γδ T cells contributing to subsets in the dermis following infection. Error bars signify SEM. Data are results from more than three (A and B) or more than two independent experiments (C and D) (n = 3–15 mice/group). *p < 0.02, **p < 0.0001, two-tailed, unpaired Student t test. N.D., not done.

FIGURE 3.

Contribution of adult BM-derived and perinatal thymus-derived γδ T cells to the dermal response. CD45.2+ TCRδ-GFP mice were irradiated and reconstituted with BM or with BM plus pThy (BMpThy). (A) Graphs show the total number of γδ T cells >8 wk following reconstitution in the dermis and skin dLN. Geometric mean is shown. (B) Contribution of BM-derived and pThy-derived γδ T cells in the dermis and LN. Geometric mean is shown. (C and D) BMpThy chimeras were infected with VV on the ear >8 wk following reconstitution. (C) CCR6 and CD27 profiles of γδ T cells in the skin dLN at day 0 and CCR6 and CD103 profiles of γδ T cells in the dermis at day 0 and 7 dpi. Numbers denote the percentage of that population within the indicated quadrant. (D) The number of BM- and pThy-derived γδ T cells contributing to subsets in the dermis following infection. Error bars signify SEM. Data are results from more than three (A and B) or more than two independent experiments (C and D) (n = 3–15 mice/group). *p < 0.02, **p < 0.0001, two-tailed, unpaired Student t test. N.D., not done.

Close modal

γδ T cell populations lost as a result of irradiation were replaced by transferring pThy into irradiated hosts along with congenically marked BM cells to generate mice referred to in this article as BMpThy chimeras. This resulted in the restoration of both dermal γδ T cells (Fig. 3A, 3B) and the circulating CCR6+ population by pThy (Fig. 3C). Total γδ T cell numbers in the dermis returned to normal levels, and the phenotype of the pThy-derived dermal γδ T cells resembled that of unirradiated mice. Although pThy-derived γδ T cells contributed to all three circulating populations defined by CD27 and CCR6 staining, CD27+ cells were a relatively minor population, allowing us to generally discriminate between the resident dermal and recruited γδ T cell populations.

Using BMpThy chimeras, we confirmed that, following VV infection, adult BM-derived γδ T cells (largely CD27+ in the circulation) were transiently recruited from the circulation and contributed primarily to the CCR6CD103 DN dermal subset (Fig. 3D). In contrast, pThy-derived γδ T cells contributed mainly to the two CD103+ subsets in the dermis and their expansion during infection. As early as day 3 following infection, pThy-derived γδ T cells had expanded in the dermis, and their numbers remained elevated for a protracted period of time. In contrast, the number of BM-derived γδ T cells peaked at 7 dpi and then declined dramatically. Importantly, at a late time point the BM-derived γδ T cells were absent from the ear, whereas the pThy-derived γδ T cells plateaued at a level similar to WT mice following infection.

Several groups have established that dermal γδ T cells are fated to produce IL-17 (16, 21, 40). We found that a fraction of dermal γδ T cells in uninfected ears produced this cytokine following in vitro stimulation with PMA/ionomycin. On day 3 after VV scarification, CD103+ γδ T cells in the dermis were capable of producing IL-17 at a level similar to that of naive tissue (Fig. 4A). Although at any given time point the majority of IL-17 production was by CD103+ γδ T cells (Supplemental Fig. 4A), it was surprising that CD103 γδ T cells also produced IL-17 in the dermis. Using the float method to preserve CD27 expression, we found that all of the IL-17 production was by CD27 γδ T cells (Supplemental Fig. 4B), which we predict are derived from the circulating CCR6CD27 population (Fig. 2C). Notably, there were no granzyme B+ γδ T cells in the naive dermis at day 3 pi, but by day 7 (which is the peak for the immigrant population), the CD103 γδ T cells produced granzyme B. None of the γδ T cells in the dermis stained positive for IFN-γ at any time following VV scarification (Supplemental Fig. 4C).

FIGURE 4.

Adult BM-derived and pThy-derived γδ T cells in the dermis are functionally distinct. TCRδ-GFP mice (A) and BMpThy chimeras (B) were infected with VV on the ear and sacrificed on day 3 or 7. Ears were digested in brefeldin A, and cells were restimulated with PMA/ionomycin for 4 h at 37°C, followed by intracellular staining for cytokines and granzyme B. Representative flow plots are shown, and gates are based on unstimulated naive samples. Numbers indicate the percentage of that population in each quadrant. Data are representative of more than two independent experiments.

FIGURE 4.

Adult BM-derived and pThy-derived γδ T cells in the dermis are functionally distinct. TCRδ-GFP mice (A) and BMpThy chimeras (B) were infected with VV on the ear and sacrificed on day 3 or 7. Ears were digested in brefeldin A, and cells were restimulated with PMA/ionomycin for 4 h at 37°C, followed by intracellular staining for cytokines and granzyme B. Representative flow plots are shown, and gates are based on unstimulated naive samples. Numbers indicate the percentage of that population in each quadrant. Data are representative of more than two independent experiments.

Close modal

We repeated these intracellular staining experiments with BMpThy chimeras to determine whether the BM- and pThy-derived populations had distinct functions (Fig. 4B). At day 3 pi, there were too few BM-derived cells to be analyzed, but the pThy-derived γδ T cells readily produced IL-17. By day 7 pi, a clear distinction between the two populations could be seen, demonstrating that, in this infection model, the function of adult BM-derived γδ T cells from the circulation was to make granzyme B, whereas the dermal pThy-derived γδ T cells gave rise to a population capable of making either IL-17 or, to a lesser extent, granzyme B.

To determine whether γδ T cells contributed to inflammation and resolution of infection, we assessed tissue pathology and viral titers in WT and TCRδ−/− mice, which lack all γδ T cells. By 3 dpi, WT ears had evidence of both epidermal and cartilage necrosis, often with entire sections of the epidermis replaced by serocellular crusts (Fig. 5A). The dermis of these mice predominantly contained accumulation of intact and degenerate neutrophils along with some macrophages and fewer lymphocytes. In contrast, there was less infiltration of neutrophils and macrophages in the ears of TCRδ−/− mice, and the cartilage and epidermis remained largely intact. Comparing the histological analysis for necrosis (combined for epidermis and cartilage) and cellularity, WT mice scored significantly higher than TCRδ−/− mice (Fig. 5B). By day 7 pi, the two groups had similar pathology (data not shown), and there was no difference in viral load between the ears of WT and TCRδ−/− mice over the course of infection either by PFU assay (Fig. 5C) or by quantitative PCR (data not shown).

FIGURE 5.

Dermal γδ T cells accelerate early collateral damage and contribute to increased tissue cellularity during VV infection. Mice were infected with VV on the ear. (A) Representative histological sections of ears stained with H&E from C57BL/6 WT and TCRδ−/− mice 3 dpi (original magnification ×200). (B) Graphs show scores for necrosis (epidermis and cartilage scored separately and then combined) and cellularity. Data are mean ± SEM. (C) WT and TCRδ−/−mice were sacrificed at the indicated time points following infection, and ears were taken for plaque assay. Error bars indicate SEM. Representative histological sections stained with H&E (D) and scores (E) for BM chimeras and BMpThy chimeras 3 dpi. Data are mean ± SEM. (F) Comparison of necrosis scores for WT, TCRδ−/−, BMpThy chimeras, and BM chimeras in relation to the presence (+) or absence (−) of γδ T cell populations. In (A) and (D), arrows indicate necrotic cartilage, arrowheads indicate neutrophilic infiltrates, and asterisks indicate serocellular crusts. Data in (C) are representative of more than one independent experiment (n = 3–6 mice/group), and data in (A), (B), (D), and (E) are representative of more than two independent experiments (n = 5–6 mice/group). Scale bars, 100 μm. *p < 0.05, **p < 0.01, ***p < 0.005, two-tailed, unpaired Student t test. Circ. circulating γδ T cells; Derm., dermal γδ T cells.

FIGURE 5.

Dermal γδ T cells accelerate early collateral damage and contribute to increased tissue cellularity during VV infection. Mice were infected with VV on the ear. (A) Representative histological sections of ears stained with H&E from C57BL/6 WT and TCRδ−/− mice 3 dpi (original magnification ×200). (B) Graphs show scores for necrosis (epidermis and cartilage scored separately and then combined) and cellularity. Data are mean ± SEM. (C) WT and TCRδ−/−mice were sacrificed at the indicated time points following infection, and ears were taken for plaque assay. Error bars indicate SEM. Representative histological sections stained with H&E (D) and scores (E) for BM chimeras and BMpThy chimeras 3 dpi. Data are mean ± SEM. (F) Comparison of necrosis scores for WT, TCRδ−/−, BMpThy chimeras, and BM chimeras in relation to the presence (+) or absence (−) of γδ T cell populations. In (A) and (D), arrows indicate necrotic cartilage, arrowheads indicate neutrophilic infiltrates, and asterisks indicate serocellular crusts. Data in (C) are representative of more than one independent experiment (n = 3–6 mice/group), and data in (A), (B), (D), and (E) are representative of more than two independent experiments (n = 5–6 mice/group). Scale bars, 100 μm. *p < 0.05, **p < 0.01, ***p < 0.005, two-tailed, unpaired Student t test. Circ. circulating γδ T cells; Derm., dermal γδ T cells.

Close modal

We next compared histologic changes of BMpThy chimeras and BM chimeras to determine whether the differences between WT and TCRδ−/− mice could be assigned to the presence or absence of resident dermal γδ T cells. By and large, the BMpThy chimeras recreated the picture seen in WT mice, with high scores for necrosis and cellularity. However, the BM chimeras had mild neutrophil infiltration, little to no cartilage necrosis, and rare epidermal necrosis (Fig. 5D, 5E). It was striking to see that replacement of dermal γδ T cells in BMpThy mice recapitulated results obtained with WT mice and, similarly, that BM mice mirrored γδ T cell–null mice (Fig. 5F). Thus, the loss of the dermal CD103+ γδ T cell population appears sufficient to reduce ear inflammation and damage.

Scarification of the skin with VV was used as a vaccine to eradicate small pox infection, and the virus is now appreciated as an important vector for vaccine development and, potentially, cancer treatment (41). We used VV scarification of mouse ears to interrogate the distinct roles of recruited and resident dermal γδ T cells and to better understand their diversity. In this study, we describe the expansion, recruitment, and contraction of γδ T cells in the dermis following localized VV infection. About half of the expanded dermal γδ T cells at day 7 were composed of a unique population of CD103CCR6 γδ T cells that rapidly appeared in the dermis in response to infection. This novel population was derived primarily from circulating CD27+ γδ T cells. A portion of the immigrant γδ T cells was phenotypically similar to the CD103+ IL-17–producing dermal-resident γδ T cell population and derived from circulating CD27 γδ T cells, although some also were found in the CD103 fraction. These immigrant populations were functionally distinct and played a discrete role from the resident dermal population, which was ultimately responsible for driving increased cellularity and tissue damage at an early time point.

The number of dermal γδ T cells increased ∼10-fold following VV infection, and our data suggest that the majority of this increase can be ascribed to immigration rather than expansion of the resident population. Thus, 7 × 105 adoptively transferred γδ T cells made up 5% of the total γδ T cell population in the spleen and LN before infection (data not shown), and this donor/host γδ T cell ratio was maintained in the dermis at day 7 following infection. Furthermore, the CD103/CCR6 profile of the adoptively transferred immigrants resembled that of the host γδ T cells in the dermis at day 7 pi (Fig. 2C), suggesting that not only is the entire CD103 population recruited, but many of the CD103+ γδ T cells in the dermis may be, as well. This, in combination with the fact that we found very little in situ proliferation (Supplemental Fig. 2), leads us to conclude that the accumulation of γδ T cells in the dermis is primarily due to recruitment.

The ability of immigrant cells to diversify into all of the dermal subsets led us to ask what the contribution was of the individual populations in the circulation. Emerging evidence confirms that CD27 delineates functional γδ T cell subsets in both mice (11, 29, 30) and humans (3133). Transfer of CD27+ and CD27 circulating γδ T cells into separate hosts, followed by VV scarification, demonstrated their unique contribution to CD103 or CD103+ dermal populations, respectively. These experiments also confirmed the independent maintenance of these two populations, even in the presence of inflammation. The stability of these two subsets and the constancy of the CCR6+/CCR6 ratio within the CD27 population offer an important tool for studying γδ T cells during inflammation. Although other groups compared CD27+ and CCR6+ γδ T cells (11, 29), the circulating CCR6CD27 subset has received less attention. This latter population is uniformly RORγt+ and is distinguished from the CCR6+ population by lower levels of CD103 and CD44 (Supplemental Fig. 1A). Future studies will be needed to explore the nuances between these populations.

Although there is some disagreement concerning the radiosensitivity of γδ T cells and their reconstitution in an irradiated adult mouse (11, 16, 22), our chimera experiments revealed that, unlike the DETC population, γδ T cells in the circulation and dermis are radiosensitive. Our results confirm that transfer of pThy into an irradiated host generates bona fide CCR6+CD27 γδ T cells in the circulation and residents in the dermis. Differential kinetics and functional profiles of the BM (immigrant) and pThy (resident) γδ T cell populations in the BMpThy chimeras allowed us to ask whether they had distinct roles following infection. Determining the role of dermal γδ T cells by comparing WT and TCRδ−/− mice is complicated by the absence of normal DETC in the null mice (14), cells that are known to play a role in keratinocyte growth and survival, perhaps impacting tissue damage or recovery. Whole-body irradiation allowed us to specifically deplete the resident dermal γδ T cells, creating mice that lack this population while retaining healthy BM-derived circulating γδ T cell populations. Comparing mice specifically with or without dermal γδ T cells revealed that the dermal-resident population was responsible for the increased inflammation observed in infected WT animals (Fig. 5D, 5E).

The ability of pThy-derived dermal γδ T cells to recruit inflammatory cells, such as neutrophils, is likely due to their production of IL-17 (16, 20, 42), and recruitment of neutrophils by γδ T cells directly results in increased tissue damage (43). We demonstrated increased necrosis and cellularity of infected ears 3 d after VV infection, indicating that the resident IL-17+ γδ T cells are early responders to viral infection. In contrast, the immigrating population of CD103CCR6 DN γδ T cells, which appeared concurrently, only upregulated granzyme B expression at a later time point. Although classically associated with cytotoxicity, which was shown for γδ T cells (44), granzyme B has direct antiviral effects within cells and can cleave structural proteins when released into the extracellular matrix (45, 46). Importantly for our study, extracellular granzyme B release was shown to have remodeling activity (47), potentially facilitating migration of cells into the tissue and impacting wound repair. Considering that the absence of γδ T cells had no effect on viral load (Fig. 5C) and the number of γδ T cells within the dermis remained elevated for an extended period of time, these granzyme-producing cells may play a nontraditional role during inflammation and in the recovery process after viral clearance.

Little is known about whether γδ T cells can form “memory.” The majority of studies have examined total γδ T cell population re-expansion (48, 49) or relied on CD44 expression (33), which is now appreciated to be regulated differently in γδ T cells than in classical αβ T cells (50). When we transferred congenically marked γδ T cells, none of the immigrant γδ T cells remained in the dermis as residents after the infection was cleared (Fig. 2B). This was true for the CD27+ circulating γδ T cells characterized as CD103CCR6 DN cells in the dermis and for the CD27 circulating cells that give rise to all three populations in the dermis at day 7 pi. Similarly, in the BMpThy chimeras, BM-derived circulating γδ T cells that entered during the infection did not remain there long term (Fig. 3D). This indicates that inflammation is not sufficient to drive the long-term skin residence of γδ T cells that enter the skin from the circulation. In contrast, it was observed that, after oral Listeria monocytogenes infection, a population of γδ T cells with memory-like function develops and persists in intestinal tissues (51). However, generating resident dermal γδ T cells requires a neonatal population (or their precursors), derived in our case from the transfer of pThy into irradiated hosts.

The presence of pThy-derived γδ T cells in the dermis was required to increase cellularity near the site of infection and to amplify the overall potency of the immune response, as demonstrated by increased tissue damage in WT and BMpThy mice. Although the complete absence of γδ T cells did not translate into increased viral load in this or related systems (52, 53), other models showed that mice lacking γδ T cells are more susceptible to bacterial infection (54) and have a decreased capacity for wound healing (13). Together, the data indicate that γδ T cells are a dynamic population with a range of functions but that they might not respond equally to all inflammatory cues. Importantly, because there is no DETC counterpart in human skin (55), it has become increasingly important to isolate the relevance of γδ T cells in the dermis in models relevant to human disease. Our study using the adoptive transfer of circulating γδ T cells into normal hosts and the creation of chimeras specifically lacking a dermal-resident population will be useful for interrogating γδ T cell subsets and furthering our understanding of the dermal immune network.

We thank the University of Washington Histology and Imaging Core for preparation of histological sections, Dr. E. Gray for critical discussion and technical advice, and Dr. P. Fink for review of the manuscript. We also thank Dr. J. Turner (University of Chicago, Chicago, IL) for the TCRδ-H2BeGFP mice originally derived by I. Prinz.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • BM

    bone marrow

  •  
  • DETC

    dendritic epidermal T cell

  •  
  • dLN

    draining lymph node

  •  
  • DN

    double negative

  •  
  • dpi, d postinfection LN

    lymph node

  •  
  • pi

    postinfection

  •  
  • pThy

    perinatal thymocyte

  •  
  • VV

    vaccinia virus, WT, wild-type.

1
Heath
W. R.
,
Carbone
F. R.
.
2013
.
The skin-resident and migratory immune system in steady state and memory: innate lymphocytes, dendritic cells and T cells.
Nat. Immunol.
14
:
978
985
.
2
Tong
P. L.
,
Roediger
B.
,
Kolesnikoff
N.
,
Biro
M.
,
Tay
S. S.
,
Jain
R.
,
Shaw
L. E.
,
Grimbaldeston
M. A.
,
Weninger
W.
.
2015
.
The skin immune atlas: three-dimensional analysis of cutaneous leukocyte subsets by multiphoton microscopy.
J. Invest. Dermatol.
135
:
84
93
.
3
Zaid
A.
,
Mackay
L. K.
,
Rahimpour
A.
,
Braun
A.
,
Veldhoen
M.
,
Carbone
F. R.
,
Manton
J. H.
,
Heath
W. R.
,
Mueller
S. N.
.
2014
.
Persistence of skin-resident memory T cells within an epidermal niche.
Proc. Natl. Acad. Sci. USA
111
:
5307
5312
.
4
Wakim
L. M.
,
Gupta
N.
,
Mintern
J. D.
,
Villadangos
J. A.
.
2013
.
Enhanced survival of lung tissue-resident memory CD8⁺ T cells during infection with influenza virus due to selective expression of IFITM3.
Nat. Immunol.
14
:
238
245
.
5
Wakim
L. M.
,
Woodward-Davis
A.
,
Bevan
M. J.
.
2010
.
Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence.
Proc. Natl. Acad. Sci. USA
107
:
17872
17879
.
6
Gebhardt
T.
,
Wakim
L. M.
,
Eidsmo
L.
,
Reading
P. C.
,
Heath
W. R.
,
Carbone
F. R.
.
2009
.
Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.
Nat. Immunol.
10
:
524
530
.
7
Mackay
L. K.
,
Stock
A. T.
,
Ma
J. Z.
,
Jones
C. M.
,
Kent
S. J.
,
Mueller
S. N.
,
Heath
W. R.
,
Carbone
F. R.
,
Gebhardt
T.
.
2012
.
Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation.
Proc. Natl. Acad. Sci. USA
109
:
7037
7042
.
8
Jiang
X.
,
Clark
R. A.
,
Liu
L.
,
Wagers
A. J.
,
Fuhlbrigge
R. C.
,
Kupper
T. S.
.
2012
.
Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.
Nature
483
:
227
231
.
9
Shibata
K.
2012
.
Close link between development and function of γ-δ T cells.
Microbiol. Immunol.
56
:
217
227
.
10
Bandeira
A.
,
Mota-Santos
T.
,
Itohara
S.
,
Degermann
S.
,
Heusser
C.
,
Tonegawa
S.
,
Coutinho
A.
.
1990
.
Localization of γ/δ T cells to the intestinal epithelium is independent of normal microbial colonization.
J. Exp. Med.
172
:
239
244
.
11
Haas
J. D.
,
Ravens
S.
,
Düber
S.
,
Sandrock
I.
,
Oberdörfer
L.
,
Kashani
E.
,
Chennupati
V.
,
Föhse
L.
,
Naumann
R.
,
Weiss
S.
, et al
.
2012
.
Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave.
Immunity
37
:
48
59
.
12
Allison
J. P.
,
Havran
W. L.
.
1991
.
The immunobiology of T cells with invariant γ δ antigen receptors.
Annu. Rev. Immunol.
9
:
679
705
.
13
Havran
W. L.
,
Jameson
J. M.
.
2010
.
Epidermal T cells and wound healing.
J. Immunol.
184
:
5423
5428
.
14
Jameson
J. M.
,
Cauvi
G.
,
Witherden
D. A.
,
Havran
W. L.
.
2004
.
A keratinocyte-responsive γ δ TCR is necessary for dendritic epidermal T cell activation by damaged keratinocytes and maintenance in the epidermis.
J. Immunol.
172
:
3573
3579
.
15
Macleod
A. S.
,
Havran
W. L.
.
2011
.
Functions of skin-resident γδ T cells.
Cell. Mol. Life Sci.
68
:
2399
2408
.
16
Sumaria
N.
,
Roediger
B.
,
Ng
L. G.
,
Qin
J.
,
Pinto
R.
,
Cavanagh
L. L.
,
Shklovskaya
E.
,
Fazekas de St Groth
B.
,
Triccas
J. A.
,
Weninger
W.
.
2011
.
Cutaneous immunosurveillance by self-renewing dermal gammadelta T cells.
J. Exp. Med.
208
:
505
518
.
17
Holtmeier
W.
,
Pfänder
M.
,
Hennemann
A.
,
Zollner
T. M.
,
Kaufmann
R.
,
Caspary
W. F.
.
2001
.
The TCR-δ repertoire in normal human skin is restricted and distinct from the TCR-δ repertoire in the peripheral blood.
J. Invest. Dermatol.
116
:
275
280
.
18
Maher
B. M.
,
Mulcahy
M. E.
,
Murphy
A. G.
,
Wilk
M.
,
O’Keeffe
K. M.
,
Geoghegan
J. A.
,
Lavelle
E. C.
,
McLoughlin
R. M.
.
2013
.
Nlrp-3-driven interleukin 17 production by γδT cells controls infection outcomes during Staphylococcus aureus surgical site infection.
Infect. Immun.
81
:
4478
4489
.
19
Byamba
D.
,
Kim
Y.
,
Kim
D.-S.
,
Kim
T.-G.
,
Jee
H.
,
Kim
S. H.
,
Park
T.-Y.
,
Yang
S.-H.
,
Lee
S.-K.
,
Lee
M.-G.
.
2014
.
Skin-penetrating methotrexate alleviates imiquimod-induced psoriasiform dermatitis via decreasing IL-17-producing γ δ T cells.
Exp. Dermatol.
23
:
492
496
.
20
Gray
E. E.
,
Ramírez-Valle
F.
,
Xu
Y.
,
Wu
S.
,
Wu
Z.
,
Karjalainen
K. E.
,
Cyster
J. G.
.
2013
.
Deficiency in IL-17-committed Vγ4(+) γδ T cells in a spontaneous Sox13-mutant CD45.1(+) congenic mouse substrain provides protection from dermatitis.
Nat. Immunol.
14
:
584
592
.
21
Cai
Y.
,
Shen
X.
,
Ding
C.
,
Qi
C.
,
Li
K.
,
Li
X.
,
Jala
V. R.
,
Zhang
H.-G.
,
Wang
T.
,
Zheng
J.
,
Yan
J.
.
2011
.
Pivotal role of dermal IL-17-producing γδ T cells in skin inflammation.
Immunity
35
:
596
610
.
22
Gray
E. E.
,
Suzuki
K.
,
Cyster
J. G.
.
2011
.
Cutting edge: Identification of a motile IL-17-producing gammadelta T cell population in the dermis.
J. Immunol.
186
:
6091
6095
.
23
Haas
J. D.
,
González
F. H. M.
,
Schmitz
S.
,
Chennupati
V.
,
Föhse
L.
,
Kremmer
E.
,
Förster
R.
,
Prinz
I.
.
2009
.
CCR6 and NK1.1 distinguish between IL-17A and IFN-γ-producing gammadelta effector T cells.
Eur. J. Immunol.
39
:
3488
3497
.
24
Maloy
K. J.
,
Odermatt
B.
,
Hengartner
H.
,
Zinkernagel
R. M.
.
1998
.
Interferon γ-producing gammadelta T cell-dependent antibody isotype switching in the absence of germinal center formation during virus infection.
Proc. Natl. Acad. Sci. USA
95
:
1160
1165
.
25
Selin
L. K.
,
Santolucito
P. A.
,
Pinto
A. K.
,
Szomolanyi-Tsuda
E.
,
Welsh
R. M.
.
2001
.
Innate immunity to viruses: control of vaccinia virus infection by γ δ T cells.
J. Immunol.
166
:
6784
6794
.
26
Mann
E. R.
,
McCarthy
N. E.
,
Peake
S. T. C.
,
Milestone
A. N.
,
Al-Hassi
H. O.
,
Bernardo
D.
,
Tee
C. T.
,
Landy
J.
,
Pitcher
M. C.
,
Cochrane
S. A.
, et al
.
2012
.
Skin- and gut-homing molecules on human circulating γδ T cells and their dysregulation in inflammatory bowel disease.
Clin. Exp. Immunol.
170
:
122
130
.
27
Argentati
K.
,
Re
F.
,
Serresi
S.
,
Tucci
M. G.
,
Bartozzi
B.
,
Bernardini
G.
,
Provinciali
M.
.
2003
.
Reduced number and impaired function of circulating γ δ T cells in patients with cutaneous primary melanoma.
J. Invest. Dermatol.
120
:
829
834
.
28
Laggner
U.
,
Di Meglio
P.
,
Perera
G. K.
,
Hundhausen
C.
,
Lacy
K. E.
,
Ali
N.
,
Smith
C. H.
,
Hayday
A. C.
,
Nickoloff
B. J.
,
Nestle
F. O.
.
2011
.
Identification of a novel proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis.
J. Infect. Dis.
187
:
2783
2793
.
29
Schmolka
N.
,
Serre
K.
,
Grosso
A. R.
,
Rei
M.
,
Pennington
D. J.
,
Gomes
A. Q.
,
Silva-Santos
B.
.
2013
.
Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory γδ T cell subsets.
Nat. Immunol.
14
:
1093
1100
.
30
Ribot
J. C.
,
deBarros
A.
,
Pang
D. J.
,
Neves
J. F.
,
Peperzak
V.
,
Roberts
S. J.
,
Girardi
M.
,
Borst
J.
,
Hayday
A. C.
,
Pennington
D. J.
,
Silva-Santos
B.
.
2009
.
CD27 is a thymic determinant of the balance between interferon-γ- and interleukin 17-producing gammadelta T cell subsets.
Nat. Immunol.
10
:
427
436
.
31
DeBarros
A.
,
Chaves-Ferreira
M.
,
d’Orey
F.
,
Ribot
J. C.
,
Silva-Santos
B.
.
2011
.
CD70-CD27 interactions provide survival and proliferative signals that regulate T cell receptor-driven activation of human γδ peripheral blood lymphocytes.
Eur. J. Immunol.
41
:
195
201
.
32
Caccamo
N.
,
La Mendola
C.
,
Orlando
V.
,
Meraviglia
S.
,
Todaro
M.
,
Stassi
G.
,
Sireci
G.
,
Fournié
J. J.
,
Dieli
F.
.
2011
.
Differentiation, phenotype, and function of interleukin-17-producing human Vγ9Vδ2 T cells.
Blood
118
:
129
138
.
33
Dieli
F.
,
Poccia
F.
,
Lipp
M.
,
Sireci
G.
,
Caccamo
N.
,
Di Sano
C.
,
Salerno
A.
.
2003
.
Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites.
J. Exp. Med.
198
:
391
397
.
34
Wang
T.
,
Scully
E.
,
Yin
Z.
,
Kim
J. H.
,
Wang
S.
,
Yan
J.
,
Mamula
M.
,
Anderson
J. F.
,
Craft
J.
,
Fikrig
E.
.
2003
.
IFN-γ-producing γ δ T cells help control murine West Nile virus infection.
J. Immunol.
171
:
2524
2531
.
35
Pennington
D. J.
,
Silva-Santos
B.
,
Shires
J.
,
Theodoridis
E.
,
Pollitt
C.
,
Wise
E. L.
,
Tigelaar
R. E.
,
Owen
M. J.
,
Hayday
A. C.
.
2003
.
The inter-relatedness and interdependence of mouse T cell receptor gammadelta+ and alphabeta+ cells.
Nat. Immunol.
4
:
991
998
.
36
Prinz
I.
,
Sansoni
A.
,
Kissenpfennig
A.
,
Ardouin
L.
,
Malissen
M.
,
Malissen
B.
.
2006
.
Visualization of the earliest steps of gammadelta T cell development in the adult thymus.
Nat. Immunol.
7
:
995
1003
.
37
Liu
L.
,
Fuhlbrigge
R. C.
,
Karibian
K.
,
Tian
T.
,
Kupper
T. S.
.
2006
.
Dynamic programming of CD8+ T cell trafficking after live viral immunization.
Immunity
25
:
511
520
.
38
Heilig
J. S.
,
Tonegawa
S.
.
1986
.
Diversity of murine gamma genes and expression in fetal and adult T lymphocytes.
Nature
322
:
836
840
.
39
Tscharke
D. C.
,
Smith
G. L.
.
1999
.
A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae.
J. Gen. Virol.
80
:
2751
2755
.
40
Sutton
C. E.
,
Lalor
S. J.
,
Sweeney
C. M.
,
Brereton
C. F.
,
Lavelle
E. C.
,
Mills
K. H. G.
.
2009
.
Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity.
Immunity
31
:
331
341
.
41
Veradi
P. H.
,
Titong
A.
,
Hagen
C. J.
.
2012
.
A vaccinia virus renaissance: new vaccine and immunotherpeutic uses after smallpox eradication.
Hu. Vac. Immuno.
8
:
961
970
.
42
Shibata
K.
,
Yamada
H.
,
Hara
H.
,
Kishihara
K.
,
Yoshikai
Y.
.
2007
.
Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production.
J. Immunol.
178
:
4466
4472
.
43
Toth
B.
,
Alexander
M.
,
Daniel
T.
,
Chaudry
I. H.
,
Hubbard
W. J.
,
Schwacha
M. G.
.
2004
.
The role of gammadelta T cells in the regulation of neutrophil-mediated tissue damage after thermal injury.
J. Leukoc. Biol.
76
:
545
552
.
44
Qin
G.
,
Mao
H.
,
Zheng
J.
,
Sia
S. F.
,
Liu
Y.
,
Chan
P. L.
,
Lam
K.-T.
,
Peiris
J. S. M.
,
Lau
Y.-L.
,
Tu
W.
.
2009
.
Phosphoantigen-expanded human gammadelta T cells display potent cytotoxicity against monocyte-derived macrophages infected with human and avian influenza viruses.
J. Infect. Dis.
200
:
858
865
.
45
Romero
V.
,
Andrade
F.
.
2008
.
Non-apoptotic functions of granzymes.
Tissue Antigens
71
:
409
416
.
46
Andrade
F.
2010
.
Non-cytotoxic antiviral activities of granzymes in the context of the immune antiviral state.
Immunol. Rev.
235
:
128
146
.
47
Buzza
M. S.
,
Zamurs
L.
,
Sun
J.
,
Bird
C. H.
,
Smith
A. I.
,
Trapani
J. A.
,
Froelich
C. J.
,
Nice
E. C.
,
Bird
P. I.
.
2005
.
Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin.
J. Biol. Chem.
280
:
23549
23558
.
48
Shen
Y.
,
Zhou
D.
,
Qiu
L.
,
Lai
X.
,
Simon
M.
,
Shen
L.
,
Kou
Z.
,
Wang
Q.
,
Jiang
L.
,
Estep
J.
, et al
.
2002
.
Adaptive immune response of Vgamma2Vdelta2+ T cells during mycobacterial infections.
Science
295
:
2255
2258
.
49
Shao
L.
,
Huang
D.
,
Wei
H.
,
Wang
R. C.
,
Chen
C. Y.
,
Shen
L.
,
Zhang
W.
,
Jin
J.
,
Chen
Z. W.
.
2009
.
Expansion, reexpansion, and recall-like expansion of Vgamma2Vdelta2 T cells in smallpox vaccination and monkeypox virus infection.
J. Virol.
83
:
11959
11965
.
50
Wencker
M.
,
Turchinovich
G.
,
Di Marco Barros
R.
,
Deban
L.
,
Jandke
A.
,
Cope
A.
,
Hayday
A. C.
.
2014
.
Innate-like T cells straddle innate and adaptive immunity by altering antigen-receptor responsiveness.
Nat. Immunol.
15
:
80
87
.
51
Sheridan
B. S.
,
Romagnoli
P. A.
,
Pham
Q.-M.
,
Fu
H.-H.
,
Alonzo
F.
 III
,
Schubert
W.-D.
,
Freitag
N. E.
,
Lefrançois
L.
.
2013
.
γδ T cells exhibit multifunctional and protective memory in intestinal tissues.
Immunity
39
:
184
195
.
52
Kim
J.-O.
,
Cha
H.-R.
,
Kim
E.-D.
,
Kweon
M.-N.
.
2012
.
Pathological effect of IL-17A-producing TCRγδ(+) T cells in mouse genital mucosa against HSV-2 infection.
Immunol. Lett.
147
:
34
40
.
53
McCarthy
M. K.
,
Zhu
L.
,
Procario
M. C.
,
Weinberg
J. B.
.
2014
.
IL-17 contributes to neutrophil recruitment but not to control of viral replication during acute mouse adenovirus type 1 respiratory infection.
Virology
456-457
:
259
267
.
54
Hamada
S.
,
Umemura
M.
,
Shiono
T.
,
Tanaka
K.
,
Yahagi
A.
,
Begum
M. D.
,
Oshiro
K.
,
Okamoto
Y.
,
Watanabe
H.
,
Kawakami
K.
, et al
.
2008
.
IL-17A produced by gammadelta T cells plays a critical role in innate immunity against Listeria monocytogenes infection in the liver.
J. Immunol.
181
:
3456
3463
.
55
Elbe
A.
,
Foster
C. A.
,
Stingl
G.
.
1996
.
T-cell receptor α β and γ δ T cells in rat and human skin—are they equivalent?
Semin. Immunol.
8
:
341
349
.

The authors have no financial conflicts of interest.

Supplementary data